1. No category

EVALUATION OF AN LC/MS MICROFLUIDIC PLATFORM FOR QUANTIFICATION OF
INTACT MONOCLONAL ANTIBODIES
Catalin E. Doneanu1,
Brad Williams2, Paul Rainville1 and Weibin Chen1
1
Pharmaceutical Business Operations, Waters Corporation, Milford, MA 01757;
2
MS Applications, Waters Corporation, Beverly, MA 01915
METHODS
OVERVIEW
Sample Preparation
Quantification of intact mAbs was performed on the
IonKey/MS platform coupled to a high-resolution Xevo
G2-S QTOF instrument.
INTRODUCTION
routinely
monoclonal
performed
on
antibodies
(mAbs)
high-resolution
is
mass
spectrometry platforms (e.g. QTOFs, Orbitrap, FTICR
MS).
A
typical
mAb
A stock solution of Trastuzumab (Herceptin) containing
20 mg/mL was diluted with an aqueous solution of
0.1% formic acid (FA, v/v) to prepare four
concentrations covering a dynamic range of 50 fold: 1,
5, 10, and 50 ug/mL.
characterization
workflow
comprises intact mAb and reduced light/heavy chain
MS analysis, LC/MS peptide mapping, sequence variant
analysis by LC/MS and impurity (host-cell protein)
profiling by 2D-LC/MS.
In contract, mAb quantification from plasma or serum
is either performed by ligand binding assays (LBA) or
ESI-MS spectrum
Assay Reproducibility
Assay LLOQ
A
m/z = 2744.0
+54
+56
Two microliters of sample were injected on an iKey
separation device (150 um ID x 5 cm) packed with 1.7
um BEH C4 particles. Separations were performed at
80 oC to improve analyte peak shape and to reduce
carryover.
Assay Linearity
+52
+58
Table I. Peak area reproducibility across the entire dynamic range investigated. Extracted mass chromatograms were generated for the
most abundant charge state observed at m/z: 2744.0 (z= 54).
LC/MS Conditions
A significant portion of structural characterization of
biotherapeutic
RESULTS
+50
+60
Figure 4. ESI-MS spectrum of Trastuzumab (10 ug/mL). The
spectrum was obtained by averaging 10 scans.
Peak Area
Mobile phase eluents contained 0.1% FA in DI water as
Solvent A and 0.1% FA in acetonitrile as Solvent B.
Each sample was loaded at a flow rate of 5 uL/min, but
the gradient elution was performed at 2 uL/min.
Trastuzumab was eluted using a fast gradient, from 10
to 50% Eluent B in 0.5 min. Full scan MS spectra were
recorded in positive ESI (Sensitivity Mode) on a Xevo
G2-S QTOF instrument over a mass range (m/z) from
800 to 4,000 with an acquisition rate of 500 msec/
spectrum.
The total runtime of the LC/MS assay was 10 min.
B
4000
3500
3000
2500
2000
1500
1000
500
0
CONCLUSIONS
R2=0.998
 A
robust
assay
was
developed
for
quantification of intact Trastuzumab.
 The
LLOQ
of
the
assay
was
1
ug/mL
of
Trastuzumab.
 The ESI-MS response of intact Trastuzumab was
0
10
20
30
40
by mAb digestion followed by SRM assays of signature
50
60
Conc (ug/mL)
Figure 2. Calibration curve for Trastuzumab in the concentration range
of 1 to 50 ug/mL.
peptides on a tandem quadrupole mass spectrometer
fast,
[1-3]. Very few attempts have been made so far
linear over the entire dynamic range tested
(1-50 ug/mL).
 Assay reproducibility was better than 15 % at all
concentrations.
toward intact mAb quantification on a high resolution
Carryover Evaluation
C
MS instrument [4] due to the limited dynamic range
and poor sensitivity.
 Trastuzumab carryover was less than 1%.
50 pg/mL Trastuzumab
References
Carryover: (21/3720) x 100 = 0.56%
Here we evaluated the analytical capabilities of the Ion
1. Heudi O, Barteau S, Zimmer D, Schmidt J, Bill K,
Key microfluidic platform coupled to a Xevo G2-S QTOF
Lehman N, Bauer C, Kretz O, Anal Chem, 2008, 80,
instrument for the quantification of intact mAbs.
Solvent A blank (0.1% FA)
Several critical features of the quantification assay,
including
lower
limit
of
quantification
4200.
2. Liu H, Manuilov A, Chumsae C, Babineau M, Tarcsa E
(LLOQ),
Anal Biochem, 2011, 414, 147.
reproducibility, linearity, dynamic range and carryover
3. Li H, Ortiz R, Tran L, Hall M, Spahr C, Walker K,
were evaluated.
Figure 1. Replicate injections (n=5) of Trastuzumab at three concentration levels: (A) 1 ug/mL at the LLOQ level; (B) 10 ug/mL; and
C) 50 ug/mL. Extracted mass chromatograms were generated for the
most abundant charge state observed at m/z = 2744.0 (+ 54).
Xevo G2-S QTOF mass spectrometer equipped with the
Ion Key source.
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Laudemann J, Miller S, Salimi-Moosavi H, Lee JW,
Figure 3. Analyte carryover was evaluated by injecting a blank
(containing mobile phase A: 0.1% FA in DI water) following the
injection of the highest concentration standard containing 50 ug/mL
Trastuzumab. The carryover calculated from the peak area ratio was
less than 1%.
Anal Chem, 2012, 84, 1267.
4. Damen C, Rosing H, Schellens J, Beijinen J, J Pharm
Biomed Applic, 2008, 46, 449.
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